Bacterial sepsis and meningitis are the leading causes of mortality and hospitalization by infectious agents in the United States. Sepsis alone accounts for >700,000 life-threatening infections each year and over 200,000 deaths, with economic losses greatly exceeding one billion annually. Extraintestinal pathogenic Escherichia coli (ExPEC) are the predominant agents of these infections, with one serotype, E. coli O18:K1:H7, emerging as the leading cause of gram-negative neonatal meningitis and the most prevalent isolate from uncomplicated cystitis in women, thus making it a useful model of ExPEC infections in general. How ExPEC breach the host's mucosa and reach the systemic concentration (bacteremia) necessary for triggering sepsis and meningitis is not understood, although cell-surface modification with sialic acid is an important determinant. In this continuation application, we propose to complete our studies to determine how the sialic and polysialic acid virulence factors are synthesized, and then to identify other gene products required for systemic disease using a functional genomic approach. Specific Aim 1 will complete the analysis of sialic acid biosynthesis and the regulation of sialate metabolism. Preliminary data indicate that the synthesis of N-acetylmannosamine (ManNAc), the first committed step in sialate biosynthesis, does not involve ManNAc 6-phosphate. X-ray crystallography and NMR spectroscopy will complement the objectives of this aim. In Specific Aim 2, the structural basis for PSA biosynthesis will be determined by analyzing chimeric polysialyltransferases (polySTs) and using site-directed mutagenesis to understand catalytic mechanism. Our preliminary data indicate that these important enzymes are structurally and functionally distinct from mammalian polySTs, suggesting wide therapeutic potential for specific polyST inhibitors. Specific Aim 3 will apply signature-tagged mutagenesis to identify disease traits that are either distinct from PSA or function in concert with this capsule to define ExPEC virulence. Our objective in this aim is to establish the minimal systemic ExPEC "pathotype". The proposed studies have direct application to the development of new therapeutic approaches by identifying novel targets for non-antibiotic drug or vaccine design. New approaches are urgently needed to prevent or treat ExPEC infections in the rapidly aging US population, the very young and the increasingly large number of immunocompromised patients.